A miniaturized wash-free microfluidic assay for electrical impedance-based assessment of red blood cell-mediated microvascular occlusion

The production of HbS - an abnormal hemoglobin (Hb) - in sickle cell disease (SCD) results in poorly deformable red blood cells (RBCs) that are prone to microcapillary occlusion, causing tissue ischemia and organ damage. Novel treatments, including gene therapy, may reduce SCD morbidity, but methods to functionally evaluate RBCs remain limited. Previously, we presented the microfluidic impedance red cell assay (MIRCA) for rapid assessment of RBC deformability, employing electrical impedance-based readout to measure RBC occlusion of progressively narrowing micropillar openings. We describe herein the design, development, validation, and clinical utility of the next-generation MIRCA assay, featuring enhanced portability, rapidity, and usability. It incorporates a miniaturized impedance analyzer and features a simplified wash-free operation that yields an occlusion index (OI) within 15 min as a new metric for RBC occlusion. We show a correlation between OI and percent fetal hemoglobin (%HbF), other laboratory biomarkers of RBC hemolysis, and SCD severity. To demonstrate the assay's versatility, we tested RBC samples from treatment-naïve SCD patients in Uganda that yielded OI levels similar to those from hydroxyurea (HU)-treated patients in the U.S., highlighting the role of %HbF in protecting against microcapillary occlusion independent of other pharmacological effects. The MIRCA assay could also identify a subset of HU-treated patients with high occlusion risks, suggesting that they may require treatment adjustments including a second-line therapy to improve their outcomes. This work demonstrates the potential of the MIRCA assay for accelerated evaluation of RBC health, function, and therapeutic effect in an ex vivo model of the microcapillary networks.